Effect of heat treatment on Cronobacter spp. in reconstituted, dried infant formula: preparation guidelines for manufacturers

Aim:  To explore safe guidelines for manufacturers and consumers to prepare, handle and store dry infant formula (DIF) to protect infants against Cronobacter spp.
Methods and Results:  Selected strains (2.45, FSM 293, ATCC-12868, FSM-271) screened from 68 strains of Cronobacter spp . were used to study growth and survival in commercial DIF. Prototype growth patterns in Enterobacteriaceae enrichment broth (EEB) containing a cocktail comprised of ATCC 12868, ATCC 29004, ATCC 29544 and ATCC 51329 showed a rapid increase in cell count (2·0 log₁₀ to 6·2 log₁₀ CFU ml⁻¹). Infant formula provided a better protective environment for the cells of Cronobacter strains than did buffered peptone water . Experiments on survival in inoculated (10⁴–10⁶ CFU ml⁻¹) reconstituted infant formula (RIF), preparation temperature, the effect of preparation volume (one-serving or two-serving) and effect of storage at room temperature for up to 10 h provided information to develop consumer guidelines for DIF preparation and handling.
Conclusions:  Reconstituted DIF in water at >70°C in larger volumes, minimizing storage time before feeding and storing unused reconstituted formulate at <4°C, may reduce the risk of Cronobacter infection in infants.
Significance and Impact of the Study:  Meningitis, necrotizing enterocolitis and bacteremia in premature babies has been linked to contaminated milk powder and DIF; better handling practices may improve the safety of these foods for neonates.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Cronobacter ('Enterobacter sakazakii'): current status and future prospects

The genus Cronobacter accommodates the 16 biogroups of the emerging opportunistic pathogen known formerly as Enterobacter sakazakii . Cronobacter spp. are occasional contaminants of milk powder and, consequently, powdered infant formula and represent a significant health risk to neonates. This review presents current knowledge of the food safety aspects of C ronobacter , particularly in infant formula milk powder. Sources of contamination, ecology, disease characteristics and risk management strategies are discussed. Future directions for research are indicated, with a particular focus on the management of this increasingly important bacterium in the production environment.     

Effect of environmental stresses on the sensitivity of Enterobacter sakazakii in powdered infant milk formula to gamma radiation

Aim:  To evaluate the effect of starvation, heat, cold, acid, alkaline, chlorine and ethanol stresses on the resistance of Enterobacter sakazakii in powdered infant milk formula (PIMF) towards gamma radiation.
Methods and Results:  Stressed cells of E. sakazakii ATCC 51329 and four other food isolate strains were mixed individually with PIMF, kept overnight at room temperature, and then exposed to gamma radiation up to 7·5 kGy. The D ₁₀-values were determined using linear regression and for the stressed E. sakazakii strains these values ranged from 0·82 to 1·95 kGy.
Conclusions:  Environmental stresses did not significantly change the sensitivity of most E. sakazakii strains to ionizing radiation.
Significance and Impact of the Study:  Data obtained established that most forms of environmental stress are unlikely to significantly enhance the resistance of E. sakazakii strains to lethal, low dose irradiation treatment.     

Rapid inactivation of <Emphasis Type="Italic">Cronobacter sakazakii</Emphasis> on copper alloys following periods of desiccation stress

Cronobacter spp. have been identified as the causative agent in meningitis and necrotizing enterocolitis in premature infants which can be linked to the bacterium's desiccation resistance and persistence in powdered infant formula. In this study we examined the efficacy of copper cast alloys in contact killing of Cronobacter sakazakii following periods of desiccation stress. Cronobacter sakazakii cells suspended in Tryptic Soy Broth (TSB) were killed within 10 min while kept moist on 99.9% copper alloys and within 1 min of drying on 99.9% copper alloys. Survival times were unchanged after cells suspended in TSB were desiccated for 33 days. Cronobacter sakazakii cells suspended in infant formula were killed within 30 min under moist conditions and within 3 min of drying on 99.9% copper alloys. However, when desiccated in infant formula for 45 days, survival times decreased to 10 and 1 min in moist and dry conditions, respectively. In contrast, no decrease in viable cells was noted on stainless steel surfaces under the experimental conditions employed in this study. Cronobacter sakazakii was rapidly killed on copper alloys under all testing conditions of this study indicating that desiccation and copper ion resistance do not prolong survival. These results could have important implications for the utilization of copper in the production and storage of powdered infant formula.     

Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia and meningitis, predominantly in neonates. Infection in these vulnerable infants has been linked to the consumption of contaminated powdered infant formula (PIF). Considerable research has been undertaken on this organism in the past number of years which has enhanced our understanding of this neonatal pathogen leading to improvements in its control within the PIF production environment. The taxonomy of the organism resulted in the recognition of a new genus, Cronobacter, which consists of seven species. This paper presents an up-to-date review of our current knowledge of Cronobacter species. Taxonomy, genome sequencing, current detection protocols and epidemiology are all discussed. In addition, consideration is given to the control of this organism in the manufacturing environment, as a first step towards reducing the occurrence of this pathogen in PIF.     

Comprehensive approaches to molecular biomarker discovery for detection and identification of Cronobacter spp. (Enterobacter sakazakii) and Salmonella spp

Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.     

Complete genome of temperate phage ENT39118 from Cronobacter sakazakii

Cronobacter sakazakii infection is particularly harmful to infants, and putative virulence factors of prophage origin have been identified in C. sakazakii. In this study, the phage ENT39118 was isolated from wild-type C. sakazakii; it belongs to the family Siphoviridae. The genomic sequence of phage ENT39118 was composed of circular double-stranded DNA with a length of 39,012 bp. The sequence of ENT39118 showed weak sequence similarity to some reported regions of the prophage sequences in the C. sakazakii BAA-894 genome. To our knowledge, this is the first study of the genomic sequencing and annotation of this temperate phage, which was obtained from a C. sakazakii isolate from powdered infant formula.     

Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula

Enterobacter sakazakii may be related to outbreaks of meningitis, septicemia, and necrotizing enterocolitis, mainly in neonates. To reduce the risk of E. sakazakii in baby foods, thermal characteristics for Korean E. sakazakii isolates were determined at 52, 56, and 60 degrees C in saline solution, rehydrated powdered infant formula, and dried baby food. In saline solution, their D-values were 12-16, 3-5, and 0.9-1 min for each temperature. D-values increased to 16-20, 4-5, and 2-4 min in rehydrated infant formula and 14-17, 5-6, and 2-3 min in dried baby food. The overall calculated z-value was 6-8 for saline, 8-10 for powdered infant formula, and 9-11 for dried baby food. Thermal inactivation of E. sakazakii during rehydration of powdered infant formula was investigated by viable counts. Inactivation of cultured E. sakazakii in infant formula milk did not occur for 20 min at room temperature after rehydration with the water at 50 degrees C and their counts were reduced by about 1-2 log CFU/g at 60 degrees C and 4-6 log CFU/ml with the water at 65 and 70 degrees C. However, the thermostability of adapted E. sakazakii to the powdered infant formula increased more than two times. Considering that the levels of E. sakazakii observed in powdered infant formula have generally been 1 CFU/100 g of dry formula or less, contamination with E. sakazakii can be reduced or eliminated by rehydrating water with at least 10 degrees C higher temperature than the manufacturer-recommended 50 degrees C.     

Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice

AIMS: To determine survival and growth characteristics of Enterobacter sakazakii in infant rice cereal as affected by type of liquid used for reconstitution and storage temperature after reconstitution. METHODS AND RESULTS: A commercially manufactured dry infant rice cereal was reconstituted with water, apple juice, milk, or liquid infant formula, inoculated with a 10-strain mixture of E. sakazakii at populations of 0.27, 0.93, and 9.3 CFU ml(-1), and incubated at 4, 12, 21 or 30 degrees C for up to 72 h. Growth did not occur in cereal reconstituted with apple juice, regardless of storage temperature, or in cereal reconstituted with water, milk, or formula and stored at 4 degrees C. The lag time for growth in cereal reconstituted with water, milk, or formula was decreased as the incubation temperature (12, 21 and 30 degrees C) was increased. Upon reaching maximum populations of 7-8 log10 CFU ml(-1), in some instances populations decreased to nondetectable levels during subsequent storage which was concurrent with decreases in pH. CONCLUSIONS: Enterobacter sakazakii initially at very low populations can rapidly grow in infant rice cereal reconstituted with water, milk, or infant formula. SIGNIFICANCE AND IMPACT OF THE STUDY: Reconstituted infant rice cereal can support luxuriant growth of E. sakazakii. Reconstituted cereal that is not immediately consumed should be discarded or stored at a temperature at which E. sakazakii and other food-borne pathogens cannot grow.     

Complete genome sequence of Cronobacter sakazakii bacteriophage CR3

Due to the high risk of Cronobacter sakazakii infection in infants fed powdered milk formula and the emergence of antibiotic-resistant strains, an alternative biocontrol agent using bacteriophage is needed to control this pathogen. To further the development of such an agent, the C. sakazakii-targeting bacteriophage CR3 was isolated and its genome was completely sequenced. Here, we announce the genomic analysis results of the largest C. sakazakii phage known to date and report the major findings from the genome annotation.     

Identification and characterization of cronobacter iron acquisition systems

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.     

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

Tracing of Enterobacter sakazakii isolates in infant milk formula processing by BOX-PCR genotyping

Aims:  Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe neonatal meningitis, bacteraemia and necrotizing enterocolitis. Contaminated powder infant formulae (PIF) have been identified as one of various infection routes. In this study, E. sakazakii was monitored in the processing environment of a PIF factory to identify possible dissemination routes.
Methods and Results:  The BOX-polymerase chain reaction (PCR), a fingerprinting technique which targets the repetitive BOX sequences, was used in routine to identify points of contamination and investigate clonal persistence. Two hundred E. sakazakii isolates were collected and typed. Most (70%) showed the same fingerprint that revealed the persistence of resident E. sakazakii strains in the processing environment. This method allowed to detect contamination of some PIF by dry-blending ingredients.
Conclusions:  Environment was the major cause for contamination of PIF and facilities. Some raw materials delivered as powder were also implicated.
Significance and Impact of the Study:  Routine BOX-PCR genotyping was very useful to trace and investigate in real-time dissemination of micro-organisms in the PIF plant and to implement a series of additional control measures to reduce the risk of final product contamination by E. sakazakii .     

Microbial contamination of food products consumed by infants and babies in Korea

Aims: The objectives of this study were to investigate the microbiological safety of various foods intended for consumption by infants and babies. Methods and Results: The incidence of Cronobacter spp. and Enterobacteriaceae from powdered infant formula (PIF, n = 75) and baby soy milk (n = 10) was examined. Additionally, aerobic plate count, coliforms and the prevalence of foodborne pathogens were investigated in 230 samples from a variety of infant and baby foods, including cereal‐based follow‐up formulas (FUF), liquid FUF and other infant foods. High APCs were observed in nutrient supplements and cereal‐based FUF. Coliforms were found in 6 (2·6%) products, and Cronobacter spp. was isolated in 10 (4·4%) samples, including four PIF and six cereal‐based FUF. Bacillus cereus was detected in 48 (20·9%) samples: cereal‐based FUF items (23·0%), rice soups (20·6%), honey samples (40·0%), biscuits (40·0%) and liquid FUF (7·4%). Conclusions: New safety criteria, along with hygienic control measures and consumer education strategies, are essential to improve the microbiological safety of infant or baby foods. Significance and Impact of the Study: This study provides comprehensive information about the prevalence and level of contamination of infant and baby food products by Cronobacter spp. and other major foodborne pathogens.     

Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.     

The phenotypic and genotypic characterization of Korean isolates of Cronobacter spp. (Enterobacter sakazakii)

This study was conducted to investigate the phenotypic and genotypic characteristics of Korean isolates of Cronobacter spp. (Enterobacter sakazakii). A total of 43 Cronobacter spp., including 5 clinical isolates, 34 food isolates, 2 environmental isolates, and 2 reference strains (C. sakazakii ATCC 29004 and C. muytjensii ATCC51329) were used in this study. Korean isolates of Cronobacter spp. were divided into 11 biogroups according to their biochemical profiles and 3 genomic groups based on the analysis of their 16S rRNA gene sequences. Biogroups 1 and 2 contained the majority of isolates (n=26), most of which were contained in 16S rRNA cluster 1 (n=34). Korean isolates of Cronobacter spp. showed diverse biochemical profiles. Biogroup 1 contained C. sakazakii GIHE (Gyeonggido Research Institute of Health and Environment) 1 and 2, which were isolated from babies that exhibited symptoms of Cronobacter spp. infection such as gastroenteritis, sepsis, and meningitis. Our finding revealed that Biogroup 1, C. sakazakii, is more prevalent and may be a more pathogenic biogroup than other biogroups, but the pathogenic biogroup was not represented clearly among the 11 biogroups tested in this study. Thus, all biogroups of Cronobacter spp. were recognized as pathogenic bacteria, and the absence of Cronobacter spp. in infant foods should be constantly regulated to prevent food poisoning and infection caused by Cronobacter spp.     

Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula

Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.